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Type I polyketides are natural products with diverse functions that are important for medical and agricultural applications. Manipulation of large biosynthetic gene clusters containing type I polyketide synthases (PKS) for heterologous expression is difficult due to the existence of conservative sequences of PKS in multiple modules. Red/ET mediated recombination has permitted rapid manipulation of large fragments; however, it requires insertion of antibiotic selection marker in the cassette, raising the problem of interference of expression by leaving "scar" sequence. Here, we report a method for precise seamless stitching of large polyketide biosynthetic gene cluster using a 48.4kb fragment containing type I PKS involved in fostriecin biosynthesis as an example. rpsL counter-selection was used to assist seamless stitching of large fragments, where we have overcome both the size limitations and the restriction on endonuclease sites during the Red/ET recombination. The compatibility and stability of the co-existing vectors (p184 and pMT) which respectively accommodate 16kb and 32.4kb inserted fragments were demonstrated. The procedure described here is efficient for manipulation of large DNA fragments for heterologous expression. Copyright © 2014 Elsevier B.V. All rights reserved.

Citation

Chun Su, Xin-Qing Zhao, Hai-Na Wang, Rong-Guo Qiu, Li Tang. Seamless stitching of biosynthetic gene cluster containing type I polyketide synthases using Red/ET mediated recombination for construction of stably co-existing plasmids. Gene. 2015 Jan 10;554(2):233-40

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PMID: 25311549

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